The epidemic of type 2 diabetes (T2D) is one of the defining medical challenges of the 21st century, with one in three Americans born in 2000 estimated to develop diabetes in their lifetime. The central initiating pathology of T2D is insulin resistance in several tissues, including liver. The normal liver maintains glycemia through gluconeogenesis and glycogenolysis. Insulin suppression of hepatic glucose production is severely impaired in diabetes, highlighting the importance of normal hepatic insulin responsiveness. All known physiological actions of insulin proceed downstream from the insulin receptor (InsR), and impairments in InsR signaling have been demonstrated in multiple models of insulin resistance. We propose to investigate two independent modes of InsR regulation with relevance to both normal physiology and insulin resistance. First, we propose to characterize a specific InsR threonine phosphorylation event that we hypothesize to be involved in lipid-induced hepatocellular insulin resistance. Briefly, hepatic insulin resistance is strongly linked t hepatocellular lipid accumulation. One potential mediator of this link is signaling through novel protein kinase C (nPKC) isoforms, especially PKCe in hepatocytes, which is activated by the lipid intermediate diacylglycerol (DAG) and has been shown to impair insulin signaling. However, the precise mechanism by which PKCe impairs insulin signaling is uncertain. In Aim 1, we will use mass spectrometry and in vitro kinase assays to validate a candidate PKCe substrate: threonine 1160 of the insulin receptor. We will then functionally characterize the importance of this interaction by performing metabolic phenotyping on a genetically modified mouse in which this threonine is knocked-in to alanine. Second, we propose to investigate the physiological significance and mechanism of action of two novel regulators of insulin receptor signaling: MARCH1 and CD82. Recently identified in a large-scale shRNA screen for negative regulators of insulin signaling, our data indicate that the E3 ubiquitin ligase MARCH1 impairs insulin signaling very proximally: at the level of the insulin receptor. Preliminary work revealed that antisense oligonucleotide blockade of MARCH1 expression is insulin-sensitizing in mice. Mechanistic studies in cells suggest that MARCH1 acts indirectly through the tetraspanin protein CD82 to alter InsR signaling. In Aim 2, we will build on these preliminary data to study the MARCH1-CD82 axis in vitro and in vivo. Using liver-specific genetic modifications, we will test whether MARCH1 requires CD82 to impair insulin signaling in vivo. In parallel, we will investigate the mechanism of this effect, hypothesizing that the MARCH1-CD82 axis alters InsR endocytic fate or steady-state plasma membrane InsR content. This proposal represents an integrated scientific approach and new learning experiences that harness techniques of physiology, cell biology, and analytical chemistry to yield novel insights into the important problem of hepatic insulin resistance.